Oligonucleotides and their analogs are widely used as research reagents. They are useful in understanding the preparation and function of many biological molecules. For example, the use of oligonucleotides and their analogs as primers in polymerase chain reactions (PCR) has given rise to an expanding commercial industry. PCR has become of significant importance in commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology is now utilized in the fields of forensics, paleontology, evolutionary studies and genetics. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for particular uses. Thus a number of chemical modifications have been introduced into oligomers to increase their usefulness in diagnostics, as research reagents and as therapeutic agents. Such modifications include those designed to increase binding to a target strand, to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
Short oligonucleotide probes used in molecular diagnostic arrays often contain albumin proteins and more specifically, covalently bound proteins such as bovine serum albumin (BSA) to enhance their binding to substrates such as nylon membranes or glass slides. Albumin generally refers to serum albumin. Albumin describes a protein or group of proteins typically found in the mammalian circulatory system. Generally, albumins are characterized by their solubility in water.
Manufacturing probes of conjugated oligonucleotides and BSA traditionally involves attaching BSA to oligonucleotides after the oligonucleotides have been synthesized and cleaved off glass beads employed in their synthesis. However, separation of excess (unconjugated) BSA which has similar molecular weight (66 kDa) to the conjugated oligonucleotide-BSA (˜72 kDa) products frequently involves high performance liquid chromatography (HPLC) purification. This process is expensive and time-consuming, resulting in a bottleneck for the probe manufacturing process. Accordingly, there is a need for an improved method of preparing conjugates of oligonucleotides and protein, more preferably albumin protein, and specifically BSA.
Prior artisans have described various conjugates and their preparation. Several prior investigations have involved conjugates of oligonucleotides and certain types of proteins. For example, in WO220544A1 entitled “Process for Preparing Peptide Derivatized Oligomeric Compounds” Manoharan et al. describe a process of using equimolar amounts of oligomeric compounds and peptide reagents in order to increase overall synthesis efficiency. This method is useful for preparing large scale amounts of peptide linked oligomeric compounds.
The process described by Manoharan et al. is not directly applicable to preparing conjugates of BSA as the stochiometry is different. In fact, the process described by Manoharan et al. has nothing to do with an enhanced synthesis method that could eliminate one or more downstream purification operations.
U.S. Pat. No. 6,210,908 entitled, “Activated Peptides and Conjugates” to Annunziato et al., describes a process that can be used to fabricate peptide conjugates for use as antigen, specific to some immunoreactive antibody. The process can enhance the yield of peptides with terminal amine-linked conjugates and decreases the reactivity of internal amine groups such that the peptide conjugate is more effective. However, this process is not particularly relevant to addressing the foregoing noted problems.
U.S. Pat. No. 5,977,299 entitled “Activated Peptides and Conjugates” describes the same process as the previously noted U.S. Pat. No. 6,210,908. And so, the '299 patent is not particularly relevant.
U.S. Pat. No. 5,767,238 entitled “Inverse Solid Phase Synthesis” is directed to a process for solution phase (homogeneous) synthesis for large chemical libraries. A large soluble polymeric group is used as support for oligonucleotide or peptide synthesis and is also subsequently used to separate products from unreacted reactants by the large size of the polymer-associated products. This process is described as improving the yield and allowing for easy purification.
It is also known to conjugate oligonucleotides with high molecular weight polyethylene glycols (PEGs), such as described in “Synthesis by High-Efficiency Liquid-Phase (HELP) Method of Oligonucleotides Conjugated with High-Molecular Weight Polyethylene Glycols (PEGs),” G. M. Bonora et al., Biological Procedures Online, vol. 1, No. 1, May 14, 1998. However, these techniques are not applicable to the objective of conjugating an oligonucleotide to a protein, and more desirably, to an albumin protein such as BSA.
Although satisfactory in certain respects, there still remains a need for a relatively simple and economical technique for preparing conjugates of protein, and more particularly an albumin protein such as BSA, and oligonucleotides that are particularly adapted for diagnostic assays.